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The production of viral vectors designed to express large and difficult to express transgenes within neurons.

Holehonnur R, Lella SK, Ho A, Luong JA, Ploski JE - Mol Brain (2015)

Bottom Line: Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons.Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains.We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner.

View Article: PubMed Central - PubMed

Affiliation: School of Behavioral and Brain Sciences and the Department of Molecular & Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA. roopa.hs@gmail.com.

ABSTRACT

Background: Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons.

Results: We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3' untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains.

Conclusions: We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express transgenes, these viral vectors likely could be useful for delivering and expressing many large or difficult to express transgenes in a neuron specific manner.

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Related in: MedlinePlus

GluN2 and GFP transgene expressionin vivomediated by lentivirus. In this experiment lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of 0.5Synapsin, CMV, 0.4αCaMKII, or 1.3αCaMKII promoters as indicated were infused into rat basal and lateral amygdala nuclei (BLA). Lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of a TRE3G promoter were infused into αCaMKII-tTA transgenic mice. Ten days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry, (IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Lentiviruses designed to express GFP from 0.5Synapsin, 0.4αCaMKII, 1.3αCaMKII, TRE3G promoters were capable of conferring GFP expression which was localized to neurons. Lentivirus designed to express GFP from a CMV promoter primarily conferred expression of GFP within glia cells. Lentiviruses designed to express Flag-GluN2A/B from either a 0.4αCaMKII or 1.3αCaMKII promoter were not capable of conferring GluN2 expression. Lentiviruses designed to express Flag-GluN2A/B from a TRE3G promoter were capable of conferring GluN2 expression as determined by IHC. Coronal sections from naïve controls were processed as a negative control for anti-Flag IHC (Negative control = coronal rat section and Negative control * = coronal mouse section), (scale bar = 50 μm).
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Fig11: GluN2 and GFP transgene expressionin vivomediated by lentivirus. In this experiment lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of 0.5Synapsin, CMV, 0.4αCaMKII, or 1.3αCaMKII promoters as indicated were infused into rat basal and lateral amygdala nuclei (BLA). Lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of a TRE3G promoter were infused into αCaMKII-tTA transgenic mice. Ten days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry, (IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Lentiviruses designed to express GFP from 0.5Synapsin, 0.4αCaMKII, 1.3αCaMKII, TRE3G promoters were capable of conferring GFP expression which was localized to neurons. Lentivirus designed to express GFP from a CMV promoter primarily conferred expression of GFP within glia cells. Lentiviruses designed to express Flag-GluN2A/B from either a 0.4αCaMKII or 1.3αCaMKII promoter were not capable of conferring GluN2 expression. Lentiviruses designed to express Flag-GluN2A/B from a TRE3G promoter were capable of conferring GluN2 expression as determined by IHC. Coronal sections from naïve controls were processed as a negative control for anti-Flag IHC (Negative control = coronal rat section and Negative control * = coronal mouse section), (scale bar = 50 μm).

Mentions: The TRE3G-GluN2A, TRE3G-GluN2B, CMV-GluN2A, CMV-GluN2B, TRE3G-GFP, and CMV-GFP lentiviruses exhibited convincing and robust transgene expression (Figure 10). Because lentivirus transduces non-neuronal cells very efficiently in vivo, the CMV-GluN2A, CMV-GluN2B lentiviruses are not suitable for in vivo use since the transgenes will be expressed robustly in non-neuronal cells. This is unfortunate considering they do function as intended. However the TRE3G-GluN2A and TRE3G-GluN2B lentiviruses function as intended too and if these viruses are used within αCaMKII-tTA transgenic mice, the GluN2 transgenes will be specifically expressed in αCaMKII positive neurons. We have found that lentiviruses transduce 293FT cells very efficiently, however lentiviruses do not transduce N2A cells well and therefore we were not able to test the lentiviruses that contained transgenes controlled by the synapsin or αCaMKII promoters in cell culture – these were tested in vivo. The 0.4αCaMKII-GluN2A, 0.4αCaMKII-GluN2B, 1.3αCaMKII-GluN2A, 1.1Syn-GluN2A, TRE3G-GluN2A, TRE3G-GluN2B, 0.4αCaMKII-GFP, 1.3αCaMKII-GFP, 0.5Syn-GFP, TRE3G-GFP, and CMV-GFP lentiviruses were infused into the basolateral amygdala and 10 days later, the animals were sacrificed and the brains were sectioned in the coronal plane and the transgene expression was examined as above. All lentiviruses were infused into rats, except the viruses containing the TRE3G promoter. Viruses containing the TRE3G promoter were infused into αCaMKII-tTA mice [38]. Each of following lentiviruses exhibited transgene expression in vivo: TRE3G-GluN2A, TRE3G-GluN2B, 0.4αCaMKII-GFP, 1.3αCaMKII-GFP, 0.5Syn-GFP, TRE3G-GFP, and CMV-GFP (Figure 11). However none of the lentiviruses which contain neuronal specific promoters were capable of conferring in vivo expression of the full length GluN2 transgenes, which is consistent with our in vitro and in vivo AAV findings.Figure 10


The production of viral vectors designed to express large and difficult to express transgenes within neurons.

Holehonnur R, Lella SK, Ho A, Luong JA, Ploski JE - Mol Brain (2015)

GluN2 and GFP transgene expressionin vivomediated by lentivirus. In this experiment lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of 0.5Synapsin, CMV, 0.4αCaMKII, or 1.3αCaMKII promoters as indicated were infused into rat basal and lateral amygdala nuclei (BLA). Lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of a TRE3G promoter were infused into αCaMKII-tTA transgenic mice. Ten days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry, (IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Lentiviruses designed to express GFP from 0.5Synapsin, 0.4αCaMKII, 1.3αCaMKII, TRE3G promoters were capable of conferring GFP expression which was localized to neurons. Lentivirus designed to express GFP from a CMV promoter primarily conferred expression of GFP within glia cells. Lentiviruses designed to express Flag-GluN2A/B from either a 0.4αCaMKII or 1.3αCaMKII promoter were not capable of conferring GluN2 expression. Lentiviruses designed to express Flag-GluN2A/B from a TRE3G promoter were capable of conferring GluN2 expression as determined by IHC. Coronal sections from naïve controls were processed as a negative control for anti-Flag IHC (Negative control = coronal rat section and Negative control * = coronal mouse section), (scale bar = 50 μm).
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Fig11: GluN2 and GFP transgene expressionin vivomediated by lentivirus. In this experiment lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of 0.5Synapsin, CMV, 0.4αCaMKII, or 1.3αCaMKII promoters as indicated were infused into rat basal and lateral amygdala nuclei (BLA). Lentiviruses designed to express Flag-GluN2A, Flag-GluN2B, or GFP under the control of a TRE3G promoter were infused into αCaMKII-tTA transgenic mice. Ten days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry, (IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Lentiviruses designed to express GFP from 0.5Synapsin, 0.4αCaMKII, 1.3αCaMKII, TRE3G promoters were capable of conferring GFP expression which was localized to neurons. Lentivirus designed to express GFP from a CMV promoter primarily conferred expression of GFP within glia cells. Lentiviruses designed to express Flag-GluN2A/B from either a 0.4αCaMKII or 1.3αCaMKII promoter were not capable of conferring GluN2 expression. Lentiviruses designed to express Flag-GluN2A/B from a TRE3G promoter were capable of conferring GluN2 expression as determined by IHC. Coronal sections from naïve controls were processed as a negative control for anti-Flag IHC (Negative control = coronal rat section and Negative control * = coronal mouse section), (scale bar = 50 μm).
Mentions: The TRE3G-GluN2A, TRE3G-GluN2B, CMV-GluN2A, CMV-GluN2B, TRE3G-GFP, and CMV-GFP lentiviruses exhibited convincing and robust transgene expression (Figure 10). Because lentivirus transduces non-neuronal cells very efficiently in vivo, the CMV-GluN2A, CMV-GluN2B lentiviruses are not suitable for in vivo use since the transgenes will be expressed robustly in non-neuronal cells. This is unfortunate considering they do function as intended. However the TRE3G-GluN2A and TRE3G-GluN2B lentiviruses function as intended too and if these viruses are used within αCaMKII-tTA transgenic mice, the GluN2 transgenes will be specifically expressed in αCaMKII positive neurons. We have found that lentiviruses transduce 293FT cells very efficiently, however lentiviruses do not transduce N2A cells well and therefore we were not able to test the lentiviruses that contained transgenes controlled by the synapsin or αCaMKII promoters in cell culture – these were tested in vivo. The 0.4αCaMKII-GluN2A, 0.4αCaMKII-GluN2B, 1.3αCaMKII-GluN2A, 1.1Syn-GluN2A, TRE3G-GluN2A, TRE3G-GluN2B, 0.4αCaMKII-GFP, 1.3αCaMKII-GFP, 0.5Syn-GFP, TRE3G-GFP, and CMV-GFP lentiviruses were infused into the basolateral amygdala and 10 days later, the animals were sacrificed and the brains were sectioned in the coronal plane and the transgene expression was examined as above. All lentiviruses were infused into rats, except the viruses containing the TRE3G promoter. Viruses containing the TRE3G promoter were infused into αCaMKII-tTA mice [38]. Each of following lentiviruses exhibited transgene expression in vivo: TRE3G-GluN2A, TRE3G-GluN2B, 0.4αCaMKII-GFP, 1.3αCaMKII-GFP, 0.5Syn-GFP, TRE3G-GFP, and CMV-GFP (Figure 11). However none of the lentiviruses which contain neuronal specific promoters were capable of conferring in vivo expression of the full length GluN2 transgenes, which is consistent with our in vitro and in vivo AAV findings.Figure 10

Bottom Line: Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons.Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains.We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner.

View Article: PubMed Central - PubMed

Affiliation: School of Behavioral and Brain Sciences and the Department of Molecular & Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA. roopa.hs@gmail.com.

ABSTRACT

Background: Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons.

Results: We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3' untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains.

Conclusions: We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express transgenes, these viral vectors likely could be useful for delivering and expressing many large or difficult to express transgenes in a neuron specific manner.

Show MeSH
Related in: MedlinePlus